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Gilles VANDERSTOCKEN

20/08/2012. Caractérisation du rôle des nucléotides extracellulaires et du récepteur purinergique P2Y 2 dans la physiopathologie des maladies pulmonaires inflammatoires. Gilles VANDERSTOCKEN. Supervisor : Dr. Didier Communi. The lung immune system.

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Gilles VANDERSTOCKEN

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  1. 20/08/2012 Caractérisation du rôle des nucléotides extracellulaires et du récepteur purinergique P2Y2 dans la physiopathologie des maladies pulmonaires inflammatoires Gilles VANDERSTOCKEN Supervisor: Dr. Didier Communi

  2. The lung immune system • 10.000-15.000 L of air isinhaledeveryday by one human • This air contains dust, pollens, bacteria, viruses, smoke, volatile chemicals, … • The lungs have to maintain a system of defence against these potentially toxic invaders : the immune system

  3. The lung immune system • First line of defencemechanisms • Second line of defencemechanisms • Innateimmunity •  Acute lunginjury •  Pulmonaryfibrosis • Adaptive immunity • Pneumonia • Asthma

  4. Differentiation of helper T cell subsets Infections Virus Fungus Bacteria IFN-g IL-12 TNF-b IgG2a IL-4IL-5 IL-13 IgE Parasites Allergens

  5. P1 and P2receptors Nucleotides Adenosine P2 P1 P2X P2Y P1 and P2 Receptors Ion channel GPCR P2Y1 P2Y2 P2Y4 P2Y6 P2Y11 P2Y12 P2Y13 P2Y14 ADP, ATP, UTP, UDP, UDP-glucose

  6. Origin and metabolism of extracellular nucleotides ATP and other nucleotides constitute “danger signals”

  7. Role of extracellular ATP in asthma ATP levels are elevated in the airways of people with asthma after allergen exposition. • If the P2 receptors are blocked (suramin) • If the ATP is neutralized (apyrase) The asthma features are suppressed (Idzko et al., Nature Medicine, 2007)  Role of P2Y2 receptor in asthma ?

  8. P2Y2Receptor in the lungs • P2Y1 and P2Y2receptorsexert a protectiveroleagainst infection of the lungs by P. aeruginosa(Geary et al., Am J Physiol Lung Cell Mol Physiol, 2005) • P2Y2 is a target receptor in cystic fibrosis therapy(Lazarowski et al., CurrOpinPharmacol, 2009)

  9. Aim of the study The aim of our work is to identify potential roles of the P2Y2 receptor in inflammatory lung diseases, using a set of in vivo models for various lung diseases in mouse. To assess these questions, we used the P2Y2WT and KO mice. • Asthma model (ovalbumin) • Acute lung injury (LPS)Pulmonaryfibrosis model (bleomycin) • Viral pneumonia model (PVM)– in collaboration with University of Liège (Laboratory of Pathology, Prof. D. Desmecht)

  10. Ovalbumin-induced ASTHMA MODEL • Asthmais a Th2 lymphocyte-associatedinflammatoryairwaydiseasecharacterized by • airway hyper-responsiveness (AHR) • increased mucus production • airwayobstruction • airwayeosinophilia

  11. MODEL OF ASTHMA Protocol Exposure of OVA sensitized C57BL/6 mice to PBS or OVA aerosols for 2 weeks Sensibilisation Exposition [IP] 10µg OVA + 1 mg Al(OH)3 vs. [IP] PBS + 1 mg Al(OH)3 [Aerosol] OVA 1% vs. PBS 30 minutes Sacrifice (Van Hove et al., J RespirCell Mol Biol, 2006)

  12. Airway inflammation is defective in ovalbumin-treated P2Y2-deficient mice  Defectiveleukocyterecruitment N = 11

  13. Defective eosinophil infiltration in the lungs of P2Y2-deficient asthmatic mice P2Y2-/- P2Y2+/+ N = 5 N = 7 Cytospin quantification Flow-cytometry quantification

  14. Level of eosinophilsrecruiters • CCL11 (eotaxin) : selectively recruits eosinophils(CCR3) by inducing their chemotaxis, and therefore, is implicated in allergic responses • IL-5 : key mediator in eosinophil activation, accumulation and maturation. N = 18

  15. Importance of VCAM-1 adhesionmolecule in asthma • VCAM-1 is a major adhesionmolecule (macrophages and eosinophils) • VCAM-1 is a 715 aminoacidtransmembraneglycoprotein • The extracellulardomain (674 a.a.) canbereleased by MMP or ADAM activities (= ECTODOMAIN SHEDDING) • Interestingly soluble VCAM-1 (sVCAM-1) isinvolved in eosinophilchemotaxis(Ueki et al., Allergy, 2009)

  16. Reduction of membrane and soluble forms of VCAM-1 in the lungs of P2Y2-/-asthmaticmice Lungsweredigestedwithcollagenase and the number of CD31/VCAM-1 cellswasquantified by flow cytometry Level of sVCAM-1 wasquantified by ELISA in the BALF N = 6 N = 11

  17. In vitro ADHESION ASSAYS on endothelialcells UTP increased macrophage adhesion on lungECsthrough P2Y2 activation and VCAM-1 regulation ECs stimulated 24h with agents Leukocytes labeled with calcein-AM Cells were co-incubated during 2h N = 4 Adherent leukocytes were counted

  18. Ovalbumin-induced ASTHMA MODEL : CONCLUSIONS (1) • Weobserved : • An inhibition of 80% of eosinophilrecruitment in the P2Y2-/-asthmaticmice • A lower expression of membrane and soluble forms of VCAM-1 Vanderstocken et al., Journal of Immunology, August 2010

  19. Ovalbumin-induced ASTHMA MODEL : CONCLUSIONS (2) 1 monthlater … (Müller et al., Allergy, September 2010) • Compared to WT animals, P2Y2-/-miceshowedreducedallergicairway inflammation with a defective migration of DCs and eosinophilstowards ATP (in vivo and in vitro) • DCs and eosinophils isolatedfromasthmaticindividualsexpressedhigherlevels of P2Y2R compared to healthycontrols

  20. LPS-induced ACUTE LUNG INJURY MODEL • To induce inflammation in the lung, P2Y2+/+ and P2Y2-/- mice were nebulized during 20 minutes with LPS (3 mg/mL) • At 6 or 24 hoursafter LPS exposure, micewerekilled and Broncho Alveolar Lavage Fluid (BALF) wascollected and analysed BALF CellscountingSupernatant Cytospin/Flow ELISA Cytometry

  21. Broncho alveolar lavage fluidanalysis N = 6 N = 9 N = 7 F4/80+ CD11b+ Gr1+ CD11c-

  22. Broncho alveolar lavage fluidanalysis : ELISA TNF-a and IL-1b are important mediators of the inflammatory response, and are involved in a variety of cellular activities, including regulation of immune cells, cell proliferation, differentiation, and apoptosis N = 9

  23. Bleomycin-induced PULMONARY FIBROSIS MODEL • Fibrosisis the formation of excessfibrous tissue in a reparativeprocess • Bleomycinis an anti-cancer agent withsideeffectssuch as pulmonaryfibrosis. It induceslunginjury via itsability to cause DNA strandbreakage and oxydant injury(Lown et al., 1977 ; Sausville et al., 1976) • This agent isused in rodents to induceexperimentallungfibrosis • Switch between inflammation and fibrosisatday 9 afterbleomycin injection and become more and more severe(Chaudhary et al., 2006) • A long period of 21 daysallows the settle of fibrosis

  24. Survival and weight loss after lung instillation with Bleomycin Intratracheal instillation of Bleomycin (0,05mg) in miceunderanesthesiaand monitored daily for survival and weightloss Day 21 : Fibrosis Day 6 : Inflammation 0,05mg

  25. Comparable airway inflammation in bleomycin-inoculated P2Y2+/+ and P2Y2-/- at day 6 N = 9 Cytospin quantification Flow-cytometry quantification N = 5 N = 8

  26. Trichrome staining of a lungatday 21 The bluestainingrepresentscollagendeposition. N = 6

  27. LPS-induced ALI and bleomycin-induced IPF : CONCLUSIONS No differenceswereobservedbetween P2Y2+/+ and P2Y2-/-animals for the LPS-induced acute lunginjury model as well as for the bleomycin-inducedpulmonaryfibrosis model

  28. PNEUMONIA VIRUS OF MICE MODEL in collaboration with Pr. Daniel Desmecht (ULg) • Pneumonia virus of mice (PVM) is a natural mouse virus affecting the respiratory tracts • Virus replication results in production of pro-inflammatory cytokines and leukocytes recruitment to the lung • This pathogen is closely related to the human respiratory syncytial virus (hRSV) • In certain case, in human newborns, early viral respiratory infections of RSV can later predispose to asthma

  29. Weight loss after lung infection with Pneumonia Virus of Mice (PVM) Micewereinoculated(underanesthesia)by intranasal instillation of 50 μl of a viral suspension containing 1000 PFU Days8 to 12 Days8 to 12 Higher mortality rate in P2Y2-/-mice infected by PVM

  30. Cellular infiltration in the lungs of PVM-infected P2Y2+/+ and P2Y2-/- mice N = 7 d10

  31. Quantification of neutrophils and macrophages, and their recruiters in the lungs A, B, C & D : N = 9 N = 12

  32. Defective infiltration of DCs, CD4+ and CD8+ Tcells in the lungs of PVM-infected P2Y2-/- mice DC markers : MHC II+ CD11c+ CD11b+ N = 5

  33. Level of dendriticcellsrecruiters • Mip-3a (CCL20) : stronglychemotactic for lymphocytes and dendriticcells • IP-10 (CXCL10) : secreted by several cell types in response to IFN-γ. Chemotactic for monocytes/macrophages, T cells, NK cells, and dendritic cells • BRAK (CXCL14) :chemotactic for monocytes and dendriticcells N = 5

  34. Detection of other inflammatory mediators in PVM-infected mice • IL-10(Treg) : anti-inflammatory cytokine, produced by monocytes and lymphocytes  no difference • IL-17 (Th17) : promotes inflammation and neutrophil recruitment and is commonly associated with allergic responses  not detected IL-12 : Polarizationof T cells to Th1 phenotype IL-6 : secreted by T cells and macrophages IFN-γ : hallmarkcytokine of Th1 cells N = 11

  35. PVM model : CONCLUSIONS Why do they die ? • Defective infiltration of DCs, and T cells in the lungs of PVM-infected P2Y2-/-mice  viral clearance ? • Decreasedlevel of IL-12 and increaselevelof IL-6 in P2Y2-/-mice ↓ Th1 response ? P2Y2+/+ P2Y2-/- d15

  36. PVM model : CONCLUSIONS • Weobserved : • a higher survival in P2Y2+/+ mice appearing from day 8 compared to the KO mice (55% vs. 13%) • an increase in the levelof neutrophils chemokines in BAL fluid of PVM-infected P2Y2-/-micecompared to WT mice (KC and MIP-2) • a decreased in the level IL-12 and an increase in the level of IL-6 in P2Y2-/-mice: ↓ Th1 • defective infiltration of DCs, CD4+ and CD8+ T cells in the lungs of PVM-infected P2Y2-/-mice

  37. The P2R family in lungdiseases

  38. GENERAL CONCLUSIONS • Innateimmunity Acute lunginjury •  Pulmonaryfibrosis • Adaptive immunity • Viral pneumonia • Asthma

  39. GENERAL CONCLUSIONS • First we demonstrated a function of P2Y2 receptor as a key mediator of Th2 immune response through the ovalbumin-induced model of asthma. Indeed, via its effect on DC activation, as well as through the regulation of endothelial and soluble VCAM-1 expression, the proinflammatory action of ATP provokes lung eosinophilia. • Second, our data support that P2Y2 receptor exerts a protective role during PVM infection through its involvement in DC and T cell infiltration and the initiation of Th1 immune response.

  40. Remerciements • Didier • Bernard • Jean-Marie • Michael • Nathalie • Larissa • Ben • Daniel • Els Des questions ?

  41. Supplemental Data

  42. P1 and P2 affinity for physiologic ligands [Physiological in plasma] : 400-700 nM [Extracellular] : 0,1 – 10 µM [Vesicles] : 150 – 1000 mM

  43. The Dual Signalling Pathway

  44. Activation cycle of G-proteins by G-protein-coupled receptors

  45. G-proteindeactivationcycle

  46. Ectonucleotidases (Field & Burnstock 2006)

  47. Assessment of lung virus yields • Titration by RT-qPCR : amounts of PVM-SH gene cDNAcopiesweremeasured • Anti-PVM : detection of antibodiesagainst PVM Anti-PVM

  48. ATP as a « danger signal » DAMPs(damage-associatedmolecularpatterns) initiate and regulate immune responses in co-operation with other danger signals, and should ideally be entities that: • (1) are constitutively present at high intracellular concentrations, • (2) are normally present at negligible extracellular concentrations, • (3) are easily released in response to injury, infection or other inflammatory stimuli, • (4) are able to activate selective and specific cellular receptors responsive over a wide range of concentrations, and • (5) are quickly degraded following their release  ATP (and Ado) meet all five of these prerequisites

  49. Adhesion and transmigration through blood vessel walls

  50. P2Y2 is a target receptor in cystic fibrosis therapy In the healthylungs, the balance between Cl-secretion and Na+ absorption represents a major mechanism of signaling to regulatemucociliary clearance activities In cystic fibrosis lungs, P2Y2 has promising perspectives as a therapeutic target to promote CaCC (Ca2+-activated Cl- channel) activity improving poor Cl- production in the airway associated with defective CFTR Airway space (Lazarowski et al, CurrOpinPharmacol, 2009)

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